The extracellular signal regulated kinase (ERK1 and ERK2) signalling pathway plays a critical role in cell proliferation. Activation of the ERK proteins either through increased expression of growth factor receptors or genetic mutations of upstream proteins is thought to be involved in the pathogenesis of many human cancers. Thus, inhibition of ERK signaling is viewed as a potential approach to prevent cancer cell proliferation. Currently, no specific inhibitors of the ERK proteins exist. Moreover, most kinase inhibitors lack specificity because they target ATP binding sites, which are well conserved among the protein kinase families. Taking advantage of recently identified ERK2 docking domains, which are reported to facilitate substrate interactions; we have used computer-aided drug design (GADD) to identify novel low molecular weight ERK inhibitors. We hypothesize that low molecular weight compounds that bind to the ERK2 docking domains will selectively block ERK interactions with substrate proteins and inhibit cell proliferation. Following CADD screening, potential compounds were selected and tested for activity in biological assays. Several compounds identified inhibited ERK-specific phosphorylation of ribosomal S6 kinase-1 (RSK-1) and the transcription factor ELK-1, both of which promote cell proliferation. In addition, active compounds showeda dose dependent reduction in the proliferation of cancer cell lines as measured by colony survival assays and bind directly to ERK2 as indicated by fluorescence spectroscopy. Aim 1of the current proposal will extend the use CADD to identify additional compounds with a high probability of binding to nine different putative binding pockets on the ERK2 docking domains. MAP kinase substrate phosphorylation assayswill be used to characterize the biological activity of the test compounds. The specific interactions between the active compounds and ERK2 will be tested by fluorescence spectroscopy and X-ray crystallography in aim 2. Lead compounds that inhibit ERK-substrate phosphorylation and bind to specific docking domains will then be tested for their ability to inhibit cancer cell proliferation in cell and animal models. At the end of this proposal, it is expected that a collection of lead compounds will be identified that block specific ERK-substrate interactions. These compounds will be appropriate for research tools and the development into novel chemotherapeutic agents aimed at treating cancers associated with elevated ERK pathway activity.